Antibiotics are chemical substances having the capacity, in a dilute solution, to kill or inhibit growth of microorganisms. Antibiotics that are sufficiently nontoxic to the host are used as chemotherapeutic agents to treat infectious diseases of humans, animals, and plants. The term was originally restricted to substances produced by microorganisms, but has been extended to include synthetic and semi-synthetic compounds of similar chemical activity.
Extensive and widespread use of antimicrobial drugs led to the emergence of resistant strains of microorganisms. These microorganisms are no longer susceptible to currently available antimicrobial drugs. In order to lower or prevent lethal infectious diseases and maintain public health, new antimicrobial agents are required. This forces researchers to pursue novel antibiotics, not yet resistant by bacteria. Antimicrobial peptides (AMPs) are part of the armament that insects have developed to fight off pathogens. Although usually cationic, the primary structures of insect AMPs vary markedly. Members of the most frequent AMP families adopt an α-helical conformation in membrane-mimetic environments (Bulet P. et al., Protein and Peptide Letters, 2005, 12, 3-11).
Insects produce antibacterial peptides, which are secreted to their hemolymph, as an innate defense against pathogenic infections (Boman, H. G. et al., Annu. Rev. Microbial., 1987, 41, 103-126). Some insect species are capable of producing 10-15 different antibiotic peptides (Hoffman, J. A., et al., FEBS Let., 1993, 325, 663-664). Each peptide has a complete different range of antibacterial action (Bulet, P. Medicine Sciences 1999. 15, 23-29).
Cecropins were first isolated from the hemolymph of Hyalophora cecropia. Cecropins are small cationic peptides consisting 29-42 amino acid residues, found in the Diptera order (genus Drosophila, Sarcophaga) and Lepidoptera order (genus Hyalophora, Manduca, Bombyx, Antheraea). It should be mentioned that a Cecropin was isolated from porcine intestine (Boman, H. G., et al. Eur. J. Biochem. 1991. 201, 23-31; Morishima, I., et al. Biochem. Physiol. 1990. 95B, 551-554; Steiner, H., et al. Nature 1981. 292, 246-248; Sun, D., et al. Biochem. Biophys. Res. Commun. 1998. 249(2), 410-415; Bulet, P. et al Immunological Reviews. 2004. 198, 169-184). The known sequences for the major Cecropins show that the N-terminal parts are strongly basic while the C-terminal regions are neutral and contain long hydrophobic stretches. In all cases the Cecropins have an amidated C-terminal residue (Boman, H. G. et al., Annu. Rev. Microbial., 1987, 41, 103-126). Cecropins secondary structure forms two amphiphatic α-helixes which are able to penetrate the bacterial membrane. This ability is followed by membrane loss of ionic gradient balance leading to bacterial death (Christensen, B. C., et al. Proc. Natl. Acad. Sci. USA. 1988 83:1670-1674; Lockey, T. D., et al. Eur. J. Biochem. 1996. 236, 263-271; Marassi, F. M., et al. Biophys. J. 1999. 77, 3152-3155; Wang, W., et al. J. Biol. Chem. 1998. 273, (42) 27438-27448).
Cecropins are very similar molecules as half the amino acid substitutions are strictly conservative. Theoretical predictions and circular dichroism spectra indicate that these peptides can form nearly perfect amphipathic α-helices with charged groups on one longitudinal side and hydrophobic side residues on the opposite side. Proteins with amphipathic helices are often associated with membranes, and this secondary structure may be of importance for the membrane-disrupting activity of the Cecropins (Boman, H. G. et al., Annu. Rev. Microbial., 1987, 41, 103-126).
The structure of different sequences of peptides of the Cecropin family shows that they represent similar types of molecules. In addition to strongly basic N-terminal region and a long hydrophobic stretch in the C-terminal half, there are other typical conserved features such as: tryptophan at position 2, the single and double lysines at positions 5, 8 and 9 and arginine at position 12. It can be concluded that there must have been strong selection pressures that have conserved certain Cecropin sequences in different types of insects throughout evolution (Boman, H. G., et al. Eur. J. Biochem. 1991. 201, 23-31).
Membrane-active peptides exhibit channel-like conductivities across planar lipid bilayer systems as well as bilayer disruption. These bilayer openings deprive the affected organisms of their transmembrane electrochemical gradients, resulting in increased water flow concomitant with cell swelling, osmolysis and cell death. Antimicrobial peptides of particular interest for pharmacological applications are those which manifest antibacterial activity, but under the same conditions, do not show hemolytic or cytotoxic effect against healthy vertebrate cells (B. Bechinger. et al. J. Membrane Biol. 1997.156, 197-211). Most antibacterial peptides have to be positively charged in order to bind to bacterial surfaces, which normally are negatively charged. Cecropins show strong antibiotic activity against a variety of Gram-negative and Gram-positive bacteria without lysing mammalian cell lines or yeast (Agerberth, B. et al. Eus. J. Biochem. 1993. 216, 623-629).
The cell killing activity of Cecropins is not mediated through specific, chiral receptor interactions. The cell lytic activity of these peptides correlates with their ability to form α-helical secondary structures in membrane environments as well as with their binding affinity to liposomes (B. Bechinger. et al. J. Membrane Biol. 1997.156, 197-211). Toxicity studies on a variety of cell types have shown that, although plant protoplasts are more sensitive to Cecropins than are animal cells, plant cells are one to two orders of magnitude less sensitive to these peptides than their bacterial pathogens (Jaynes, J. M., et al. Peptide Res. 1989. 2, 157-160; Nordeen, R. D., et al. Plant Sci. 1992. 82, 101-107).
A strong example for Cecropin advantage as antimicrobial agents can be found in Cecropin A. Cecropin A, a 37-residue peptide, is composed entirely of ordinary
L-amino acids (Steiner H., et al. Nature. 1981, 292:246-248). Cecropin A secondary structure is composed of two amphiphatic α-helixes with an identical length of bacterial plasma membrane. The primary target of this toxin is assumed to be the microbial membrane, and its antimicrobial effect is probably due to ionophore activity. When Escherichia coli bacteria were treated with Cecropin A, K+ ions inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of Cecropin A was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation (Natori, S. Nippon Rinsho. 1995. 53, 1297-1304; Okada, M., et al. Biochem. J. 1985.229, 453-458, Silvestro L. et al. Antimicrob Agents Chemother. 2000 March; 44(3): 602-607).
It should be noted that Cecropins inhibited the growth of harmful bacteria in the human intestine without affecting the growth of beneficial bacteria which are abundant in the intestines of healthy people (Mitsuhara, I., et al. Biotechnology Letters. 2001. 23, 569-573).
The use of peptides as antibiotics is not obvious due to their sensitivity to protease activity (Andrew, D., et al. Biopolymers. 1998.47, 415-433). Most Cecropins are rich with Lysine and Arginine residues, which commonly comprise part of target sequences for abundant proteases such as trypsin, Inhibitor A and Proteinase K (Gunnel DALHAMMAR et al. Eur. J. Biochem. 139, 247-252 (1984, Bland J M et al. Journal of agricultural and food chemistry 1998 v. 46 no. 12 pp. 5324-5327). Previous research has shown that Cecropins are rapidly degraded in the intracellular fluid of plants (Owens, L. D., et al. Mol. Plant Microbe Interact. 1997. 10, 525-528). Several experiments trying to express Cecropins in plants have failed probably due to sensitivity to proteolytic activity (Allefs, S. J. H. M., et al. Am. Potato J. 1995. 72, 437-445; Florack, D., et al. Transgenic Res. 1995. 4, 132-141; Hightower, R., et al. Plant Cell Rep. 1994. 13, 295-299).
The engineering of stable proteins is of great technological and economic importance, since the limited stability of proteins often severely restricts their medical and industrial application. It is therefore an object of the invention to provide novel stable peptide-based antibiotics, such as AMCP's.